Duct Tumor Research Articles

Estrogen receptor signaling regulates the expression of the breast tumor kinase in breast cancer cells

BackgroundBRK is, a non-receptor tyrosine kinase, overexpressed in approximately 85% of human invasive ductal breast tumors. It is not clear whether BRK expression correlates with breast cancer subtypes, or the expression has prognostic or diagnostic significance. Herein, we investigated the correlation of BRK with any breast cancer subtypes and clinicopathological significance of BRK expression in breast cancer.MethodsIn this study, we examined BRK expression in 120 breast tumor samples and 29 breast cancer cell lines to explore the positive correlation between BRK and the expression of ERα. We used immunohistochemistry, RT-PCR, and immunoblotting to analyse our experimental samples.ResultWe demonstrate that estrogen induces BRK gene and protein expression in ER+ breast cancer cells. Over-expression of ERα in the ER-negative breast cancer cell line increased BRK expression, and knock-down of ESR1 in MCF7 cells reduced BRK levels. Further, we provide evidence that BRK is regulated by ERα signaling and the presence of ER antagonists (tamoxifen and fulvestrant) reduce the expression of BRK in ER-positive breast cancer cells. Finally, we demonstrate that the overall survival of ER-positive breast cancer patients is poor when their cancers express high levels of BRK.ConclusionOur data indicate that BRK is a prognostic marker for ER+ breast cancers and provide a strong rationale for targeting BRK to improve patients’ survival.

Mutation profiling in the PIK3CA, TP53, and CDKN2A genes in circulating free DNA and impalpable breast lesions

Breast impalpable lesions have become a clinical dilemma because they are small, presenting a heterogeneous cellular phenotype. The aim of this study was to evaluate the mutational profile of the PIK3CA, TP53, and CDKN2A genes, comparing the mammary tissue with the respective circulating free DNA (cfDNA). The PIK3CA, TP53, and CDKN2A genes were sequenced (PCR-Sanger) in 58 women with impalpable lesions (49 malignant and 9 benign) with the respective cfDNA. The chi-square or Fisher's exact test was used to evaluate statistical significance between the clinical variables and mutational profile. A total of 51 out of 58 samples generated successful mutation profiles in both breast lesion and cfDNA. Of the 37 mutations detected, 10 (27%) and 16 (43%) mutations were detected in benign and malignant breast lesions, respectively, while 2 (5%) and 9 (24%) were found in cfDNA of women with benign and malignant lesions, respectively. The lymph node involvement with mutations in the PIK3CA in malignant lesions (P = 0.001), and the relationship between mutations in PIK3CA, comparing ductal tumors with benign lesions (P = 0.05), were statistically significant. This study detected different mutations in PIK3CA, TP53, and CDKN2A genes, which represent, in part, the heterogeneity of impalpable lesions. The results confirm that more studies should be conducted on the functional role of cfDNA in the impalpable lesions.

An autopsy case of primary extranodal NK/T cell lymphoma (extranodal NK/T-cell lymphoma) of the bile duct

We reported the case of a 50-year-old man diagnosed with extrinsic NK/T-cell lymphoma. He was initially diagnosed with locally advanced unresected pancreatic duct carcinoma and was treated with combination chemotherapy using gemcitabine and nabpaclitaxel. One month after treatment, he developed bleeding. Upper gastrointestinal endoscopy showed a deep ulcer lesion from the duodenal bulb to the inner wall of the descending section that was not observed before treatment. Coil embolization was performed, but the necrotic area widened after treatment; the patient died of disseminated intravascular coagulation after 1 week. Autopsy showed a soft white-tone lesion that extended from the ulcer wall to the gallbladder wall and around the intrahepatic bile duct. Lesions were also found in the spleen, lungs, kidney, and bone marrow, and immunohistochemistry confirmed extrinsic NK/T-cell lymphoma (extranodal NK/T-cell lymphoma, nasal type). In conclusion, histological diagnosis of NK/T-cell lymphoma is difficult at an early stage, and the clinical course often shows rapid tumor progression, particularly bleeding in the digestive organs or widespread perforation and penetration. NK/T-cell lymphoma should be ruled out in patients with bile duct and pancreatic tumors in whom tissue diagnosis via biopsy cannot be performed.

IQGAP1 Maintains Pancreatic Ductal Adenocarcinoma Clonogenic Growth and Metastasis.

IQ motif containing GTPase-activating protein 1 (IQGAP1) acts as a scaffold for aberrant mitogen-activated protein kinase (MAPK) signaling driven by KRAS mutations in pancreatic ductal adenocarcinoma (PDAC). We determined the role of IQGAP1 in clonogenic growth and metastasis in PDAC. We inhibited IQGAP1 expression using shRNA and assessed clonogenic growth, cell migration, and MAPK signaling in vitro and tumor initiation and metastasis in vivo. The efficacy of a peptide mimicking the IQGAP1 WW domain that binds and inhibits ERK1/2 was determined in vitro and in vivo. IQGAP1 loss inhibited clonogenic growth and migration of KRAS-dependent PDAC cells by disrupting MAPK signaling. In mice, IQGAP1 knockdown decreased tumor-initiating cell frequency and metastasis. WW peptide treatment inhibited clonogenic growth and in vivo tumor growth. Pancreatic ductal adenocarcinoma clonogenic growth, metastasis, and tumor initiation are dependent on MAPK signaling via IQGAP1. Treatment with a WW peptide disrupts IQGAP1 function and represents a novel targeting strategy for PDAC.

Differential diagnostic value of the expression of the transcription factor PDX-1 in neuroendocrine and non-neuroendocrine tumors of the pancreas and other organs

to comparatively analyze and assess the value of the expression of the TF PDX-1 in NETs and non-NETs of different localization and histogenetic origin. Anti-PDX-1 antibodies were used to study 528 tumors divided into 3 groups: Group 1 included 394 NETs, among them there were those of the pancreas (n=173), stomach (n=46), bowel (n=65), lung (n=40), thymus (n=8), kidney (n=6), Merkel's cell carcinomas (n=14), NETs of the breast (n=3), larynx (n=2), trachea (n=2), bladder (n=1), and metastatic NETs (n=34) of unknown primary site; Group 2 consisted of 16 tumors, of them there were paragangliomas (n=6), medullary thyroid cancers (MTC) (n=6) and adrenal pheochromocytomas (APCC) (n=4); Group 3 comprised 118 non-NETs, among them there were tumors of the pancreas (n=54), stomach (n=26), bowel (n=17), lung (n=11), breast (n=3), kidney (n=4), adrenal glands (n=2), and bladder (n=1). PDX-1 was positive in 75.1% (130/173) of pancreatic NETs, all insulinomas (50/50), gastrinomas (11/11), somatostatinomas (3/3), ACTH-producing tumors (2/2); PDX-1 was positive in the non-functioning pancreatic NETs, all PPomas (19/19), 76.1% (35/46) of NETs without the hormone detected, 50% (2/4) of calcitoninomas, and 21.1% (8/38) of silent glucagonomas. PDX-1 was positive in 32.4% (11/34) of carcinoids and 50% (6/12) of neuroendocrine carcinomas, all duodenal NETs (18/18), 90% (9/10) of rectal carcinoids and 30.8% (4/13) colonic carcinoids, 37.5% (3/8) of thymic/mediastinal carcinoids, 66.7% (4/6) of kidney carcinoids, and 37.5% (9/24) of metastatic NETs of unknown primary site. PDX-1 was negative in all carcinoids of the colon and sigmoid (0/5), ileum and jejunum (0/24), lung (0/40), trachea (0/2), larynx (0/2), Merkel's cell carcinoma (0/14), breast (0/3), bladder (0/1), as well as MTC (0/6), APCC (0/4), and paragangliomas (0/6). PDX-1-positive non-NETs included 81.8% (18/22) of adenocarcinomas (AC) and all serous cystic, mucinous cystic, intraductal and acinar cell tumors of the pancreas (4/4, 3/3, 2/2, and 3/3), 57.1% of AC (8/14) and 83.3% of signet ring cell carcinomas of the stomach (10/12), 56.2% AC of the bowel (9/17), bladder cancer (1/1). PDX-1 was negative in all anaplastic cancers (0/2) and solid pseudopapillary tumors of the pancreas (0/20), cancers of the lung (0/11), kidney (0/4), breast (0/3), and adrenal glands (0/2). The expression of PDX-1 is very specific for most digestive tract NETs and non-NETs. Pancreatic ductal and acinar cell tumors and gastric signet ring cell carcinomas are most commonly PDX-1-positive. Most tumors that do not originate from the digestive tract have a PDX-1 negative immunophenotype.

Differences in race, molecular and tumor characteristics among women diagnosed with invasive ductal and lobular breast carcinomas.

The dominant invasive breast cancer histologic subtype, ductal carcinoma, shows intrinsic subtype diversity. However, lobular breast cancers are predominantly Luminal A. Both histologic subtypes show distinct relationships with patient and tumor characteristics, but it is unclear if these associations remain after accounting for intrinsic subtype. Generalized linear models were used to estimate relative frequency differences (RFDs) and 95% confidence intervals (95% CIs) for the associations between age, race, tumor characteristics, immunohistochemistry (IHC) and RNA-based intrinsic subtype, TP53 status, and histologic subtype in the Carolina Breast Cancer Study (CBCS, n = 3,182) and The Cancer Genome Atlas (TCGA, n = 808). Relative to ductal tumors, lobular tumors were significantly more likely to be Luminal A [CBCS RNA RFD: 44.9%, 95% CI (39.6, 50.1); TCGA: RFD: 50.5%, 95% CI (43.9, 57.1)], were less frequent among young (≤ 50years) and black women, were larger in size, low grade, lessfrequently had TP53 pathway defects, and were diagnosed at later stages. These associations persisted among Luminal A tumors (n = 242). While histology is strongly associated with molecular characteristics, histologic associations with age, race, size, grade, and stage persisted after restricting to Luminal A subtype. Histology may continue to be clinically relevant among Luminal A breast cancers.

Development of a Class Prediction Model to Discriminate Pancreatic Ductal Adenocarcinoma from Pancreatic Neuroendocrine Tumor by MALDI Mass Spectrometry Imaging.

To define proteomic differences between pancreatic ductal adenocarcinoma (pDAC) and pancreatic neuroendocrine tumor (pNET) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Ninety-three pDAC and 126 pNET individual tissues are assembled in tissue microarrays and analyzed by MALDI MSI. The cohort is separated in a training (52 pDAC and 83 pNET) and validation set (41 pDAC and 43 pNET). Subsequently, a linear discriminant analysis (LDA) model based on 46 peptide ions is performed on the training set and evaluated on the validation cohort. Additionally, two liver metastases and a whole slide of pDAC are analyzed by the same LDA algorithm. Classification of pDAC and pNET by the LDA model is correct in 95% (39/41) and 100% (43/43) of patients in the validation cohort, respectively. The two liver metastases and the whole slide of pDAC are also correctly classified in agreement with the histopathological diagnosis. In the present study, a large dataset of pDAC and pNET by MALDI MSI is investigated, a class prediction model that allowed separation of both entities with high accuracy is developed, and differential peptide peaks with potential diagnostic, prognostic, and predictive values are highlighted.

Technical Note: In vivo Young's modulus mapping of pancreatic ductal adenocarcinoma during HIFU ablation using harmonic motion elastography (HME).

Noninvasive quantitative assessment of coagulated tissue during high-intensity focused ultrasound (HIFU) ablation is one of the essential steps for tumor treatment, especially in such cases as the Pancreatic Ductal Adenocarcinoma (PDA) that has low probability of diagnosis at the early stages and high probability of forming solid carcinomas resistant to chemotherapy treatment at the late stages. Harmonic motion elastography (HME) is a technique for the localized estimation of tumor stiffness. This harmonic motion imaging (HMI)-based technique is designed to map the tissue Young's modulus or stiffness noninvasively. A focused ultrasound (FUS) transducer generates an oscillating, acoustic radiation force in its focal region. The two-dimensional (2D) shear wave speed, and consequently the Young's modulus maps, is generated by tracking the radio frequency (RF) signals acquired at high frame rates. By prolonging the sonication for more than 50s using the same methodology, the 2D Young's modulus maps are reconstructed while HIFU is applied and ablation is formed on PDA murine tumors. The feasibility of this technique in measuring the regional Young's modulus was first assessed in tissue-mimicking phantoms. The contrast-to-noise ratio (CNR) was found to be higher than 11.7dB for each 2D reconstructed Young's modulus map. The mean error in this validation study was found to be equal to less than 19%. Then HME was applied on two transgenic mice with pancreatic ductal adenocarcinoma tumors. The Young's modulus median value of this tumor at the start of the HIFU application was equal to 2.1kPa while after 45s of sonication it was found to be approximately three times stiffer (6.7kPa). The HME was described herein and showed its capability of measuring tissue stiffness noninvasively by measuring the shear wave speed propagation inside the tissue and reconstructing a 2D Young's modulus map. Application of the methodology invivo and during HIFU were thus reported here for the first time.

Cholangioscopy in the digital era.

Cholangioscopy allows direct visualization and subsequent therapeutic maneuvers of the biliary ductal system. With advances in endoscopic and imaging technology, cholangioscopy has become an important modality for the diagnosis of indeterminate biliary strictures and an essential therapeutic tool for difficult to remove biliary stones. Enhanced imaging and operability of the latest generation cholangioscopes have further expanded their clinical applications to include ductal tumor ablation, gallbladder drainage, access to difficult to reach branches of the biliary tree, and biliary foreign body manipulation. In this review, we discuss the technical evolution of cholangioscopy into the digital era and review the clinical evidence supporting its use in the diagnosis and therapy of biliary tract disease.

Association of the location of pancreatic ductal adenocarcinoma (head, body, tail) with tumor stage, treatment, and survival: a population-based analysis

Background: The association between pancreatic ductal adenocarcinoma (PDAC) location (head, body, tail) and tumor stage, treatment and overall survival (OS) is unclear.Methods: Patients with PDAC diagnosed between 2005 and 2015 were included from the population-based Netherlands Cancer Registry. Patient, tumor and treatment characteristics were compared with the tumor locations. Multivariable logistic and Cox regression analyses were used.Results: Overall, 19,023 patients were included. PDAC locations were 13,451 (71%) head, 2429 (13%) body and 3143 (16%) tail. Differences were found regarding metastasized disease (head 42%, body 69%, tail 84%, p < .001), size (>4 cm: 21%, 40%, 51%, p < .001) and resection rate (17%, 4%, 7%, p < .001). For patients without metastases, median OS did not differ between head, body, tail (after resection: 16.8, 15.0, 17.3 months, without resection: 5.2, 6.1, 4.6 months, respectively). For patients with metastases, median OS differed slightly (2.6, 2.4, 1.9 months, respectively, adjusted HR body versus head 1.17 (95%CI 1.10–1.23), tail versus head 1.35 (95%CI 1.29–1.41)).Conclusions: PDAC locations in body and tail are larger, more often metastasized and less often resectable than in the pancreatic head. Whereas survival is similar after resection, survival in metastasized disease is somewhat less for PDAC in the pancreatic body and tail.

Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer

BackgroundMurine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.MethodsIntraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.ResultsMice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.ConclusionsThe current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.

Abstract B41: BP1 is an important biomarker in breast cancer

Abstract Beta protein 1 (BP1), a transcription factor (TF) we identified and cloned, is encoded by a homeobox gene called DLX4. BP1 is overexpressed in breast cancer, prostate cancer, ovarian cancer, acute myeloid leukemia, non-small cell lung cancer, and possibly other malignancies as well. Important characteristics of BP1 in breast cancer, a focus in our laboratory, include findings that: (1) BP1 is expressed in 80% of invasive ductal breast tumors, including 89% of the tumors of African American women. These data are based on both RNA and protein data. (2) BP1 expression correlates with the progression of breast tumors, from 0% in normal breast tissue to 21% in hyperplasia and 46% in ductal carcinoma in situ. (3) BP1, which maps to 17q21, can be activated by DNA amplification. (4) BP1 appears to be associated with metastasis. Forty-six cases of inflammatory breast cancer were examined; all were positive for BP1 expression. Nine cases had metastasized; lymph nodes from all nine were also BP1 positive. Moreover, BP1 activates a known trigger of metastasis, the Twist gene. (5) BP1 overexpression induces oncogene expression. BP1 activates the BCL-2 gene; high BCL-2 protein levels are associated with resistance to drug and radiation therapy. BP1 also activates VEGF and c-MYC, as well as other genes important in angiogenesis, invasion, and metastasis. (6) Cells overexpressing BP1, when injected into the fat pads of nude mice, were associated with larger and more frequent tumors than found in control mice receiving cells with low BP1. In summary, BP1 appears to confer properties on breast cancer cells that lead to a more invasive and aggressive phenotype. Since the functions of homeotic transcription factors are highly conserved, it is possible that BP1 regulates many of the same processes and genes in other malignancies in which it is active. Citation Format: Patricia E. Berg, Yan-gao Man, Samuels Simmens, Sidney W. Fu, Lucianne Cavalli, Farhad Vesuna, Saurabh Kirolikar, Arnold Schwartz. BP1 is an important biomarker in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B41.

Sentinel node biopsy after neoadjuvant chemotherapy for breast cancer with axillary node metastasis: A survey of clinical practice

A survey of breast surgeons was conducted to evaluate changes in clinical practice regarding sentinel node biopsy (SNB) among clinically node-negative patients after neoadjuvant chemotherapy for breast cancer with axillary node metastasis. We conducted two surveys among 252 members of the Korean Breast Cancer Society. The questionnaire comprised a case presentation and two associated questions. The case outlined a woman diagnosed with right breast cancer; core needle biopsy had confirmed invasive ductal carcinoma and tumor size was 4cm on imaging examination. Fine needle aspiration examination for axillary lymph node enlargement showed metastatic carcinoma. The patient underwent neoadjuvant chemotherapy and tumor size was decreased by 2cm; axillary lymph node enlargement was not observed on palpitation or imaging examination. Finally, the patient underwent breast conserving surgery. Survey recipients were asked: 1. Would you perform SNB in this patient? 2. If you perform SNB and no axillary node metastasis is seen, would you perform additional axillary lymph node dissection (ALND)? The response rate was 28.2% (71/252) and 15.1% (38/252) in 2013 and 2017, respectively. For the first question, the SNB to ALND ratio increased significantly from 54% versus 46% in 2013 to 92% versus 8% in 2017 (p<0.001). The proportion of surgeons performing no additional ALND versus additional ALND increased from 38% versus 54% in 2013 to 53% versus 37% in 2017, but did not reach statistical significance (p=0.1). These data show that application of SNB among clinically node-negative patients after neoadjuvant chemotherapy for breast cancer with node metastasis has increased among surgeons in Korea.

Upregulating MMP-1 in carcinoma-associated fibroblasts reduces the efficacy of Taxotere on breast cancer synergized by Collagen IV.

Chemotherapy is an important comprehensive treatment for breast cancer, which targets micro-environment of tumors as well as their characterisitcs. A previous microarray analysis revealed that matrix metalloproteinase (MMP)-1 was highly upregulated in carcinoma-associated fibroblasts (CAFs) prior to and following treatment with Taxotere under co-culture conditions. However, whether the chemotherapeutic effects of Taxotere were influenced by the changes in MMP-1 remained unclear. The purpose of the present study was to investigate the impact and mechanism of CAFs in regulating the efficacy of Taxotere on breast cancer cells. CAFs isolated from primary invasive ductal human breast tumors following surgical resection, were used in co-culture with MDA-MB-231 cells to simulate the tumor micro-environment. Following the addition of Taxotere, changes in MMP-1 gene and protein expression were assessed by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Proliferation, invasion and apoptosis assays revealed that when MMP-1 was upregulated in CAFs, the therapeutic efficacy of Taxotere was reduced in breast cancer cells. Chemosensitivity was significantly increased when MMP-1 expression was inhibited by GM6001. In addition, Collagen IV was upregulated in CAFs following chemotherapy and protected breast cancer cells against chemotherapeutic side effects. Collagen IV expression significantly decreased, as well as MMP-1 expression when GM6001 was added. Proliferation and invasion assays demonstrated that the exogenous addition of Collagen IV weakend the chemotherapeutic effect of Taxotere on breast tumor cells. Overall, the results revealed that in CAFs, MMP-1 synergized with Collagen IV as a key gene in regulating the chemotherapeutic effect of Taxotere on breast tumor cells and served an important role in reducing the efficacy of Taxotere on breast cancer, potentially via the transforming growth factor-β signaling pathway. These fidings provide a theoretical basis for the mechanism of CAFs in reducing the chemotherapeutic effect of Taxotere on breast cancer cells and a novel approach for enhancing the chemosensitivity of tumors.

Abstract 4480: Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration

Abstract The Iroquois homeobox 1 (IRX1) belongs to Iroquois homeobox family and plays an important role during embryonic development. Recent studies have suggested that IRX1 acts as a tumor suppressor in gastric cancer and head and neck carcinoma. The zebrafish allows easy genetic modification at a lower cost, thus being a valuable model for cancer biology. For functional annotation we established homozygous knockout mutants of Irx1a and Irx1b and followed to evaluate long-term phenotypes. Homozygous knockout zebrafish for either Irx1a or Irx1b are vital and survived long enough to see long-term phenotypes. However, majority of the homozygotes for both Irx1a /b were severely malformed, could not survive longer than 6 month, and were not fertile. Surprisingly, hyperplasia and tumorigenesis developed in homozygous Irx1a mutants in multiple organs, including bile duct, intestine, kidney, ovary, and testis where irx1 genes are expressed in adults when evaluated by in situ hybridization. Bile duct hyperplasia or tumor was the most common phenotype, occurring in half and two thirds at 6 and 12 months, respectively. Irx1b mutants also developed similar tumorigenesis but showed less prevalent phenotypes than Irx1a mutants, suggesting functional preservation and redundancy. Analysis of differential genes (Wild type vs. Irx1a/b knockout) by cRNA microarray (Agilent Zebrafish v3 GE 4X44 Microarrays) revealed 687 up- and 963 downregulated genes by Irx1a/b knockout. Significant canonical pathways were cyclins and cell cycle regulation, mitotic roles of polo-like kinase, FXR/RXR activation, estrogen-mediated S-phase entry, and cell cycle control of chromosomal replication, suggesting aberrant cell cycle regulation as an important mechanism of tumorigenesis. Real-time RT PCR validated the differential expression of cell cycle-related genes, especially genes involved in G2/M phase progression. Differential genes included CCND1, ARAF, CDKN2C, and FGFR1 among the upregulated genes, and ANAPC7, CCNA2, CCNB1, 2, HAUS1, 3, 6, CDC7, CDC20, CDCA8, FBXO5, 32, 43 and PLK3 among the downregulated genes. Overexpression by transfection decreased cell proliferation in cholangiocarcinoma cells (SNU-1196 and HuCCT1) without altering sensitivity to H2O2-induced apoptosis on annexin V flow cytometry. Western blot revealed decreased expression of CCND1, CCNE, CCNA1, CCNB1 by IRX1 expression. Flow cytometry using propidium iodide and Alexa647-anti-pHH3 showed decreased mitotic fraction by IRX1 expression. On cell cycle synchronization and release using hydroxyuria and nocodazole showed slight delay in G1-S progression and marked delay in G2-M progression and entry into G1. In conclusion, this study identified Irx1 gene as an important tumor-suppressor gene at various organs, and knockout or Irx1 leads to tumorigenesis by altering cell cycle regulation, especially at G2/M phase. Citation Format: Inhye Jung, Dawoon E. Jung, DoHee Kim, Dong Hee Kim, Yong Yoon Chung, Seung Woo Park. Irx1 gene knockout in zebrafish induces to multiorgan tumorigenesis through cell cycle aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4480.

Abstract 3338: BP1 induces an epithelial to mesenchymal transition in breast cancer cells by modulating the Twist/IL6 pathway

Abstract Background. BP1 (Beta Protein 1) belongs to the Distal-less family of homeobox genes. We have demonstrated that BP1 is activated in over 80% of invasive ductal breast tumors, where it is associated with breast cancer progression. The mechanism(s) of BP1 involvement in breast cancer progression, invasion and metastasis are still not known. Homeobox genes contribute to the epithelial to mesenchymal transition (EMT). During EMT, epithelial cells acquire mesenchymal features which lead to motility, invasiveness and resistance to apoptosis. EMT is also characterized by changes in apico-basal polarity and a dramatic remodeling of the cytoskeleton. During progression toward metastasis, cancer cells acquire a mesenchymal gene expression phenotype and increased motility. This transition allows the tumor cells to metastasize and establish secondary tumors at distant sites. One of the drivers of the EMT is the transcription factor Twist. Twist is a member of highly conserved family of basic helix-loop-helix transcription factors and is involved in the specification and differentiation of mesenchymal tissue in embryos. Twist overexpression can induce EMT, generate cancer stem cells, and promote metastasis in vivo. We hypothesized that BP1 might promote cancer metastasis and invasiveness mediated though Twist and the EMT. Methods. We generated BP1 overexpressing cell lines, MCF-7/BP1 and HS578T/BP1, along with the associated vector controls. Expression of BP1 was tested by both protein and transcript levels by Western blotting and qRT-PCR, respectively. BP1 binding to Twist was assayed by chromatin immunoprecipitation. Confocal microscopy was used to localize BP1 expression and Boyden chamber assays were employed to assess the migratory and invasive ability of these cells. ELISA assays were employed to study IL6 expression in BP1 expressing cells. Results. BP1 expression led to an increase in Twist expression. Mechanistically, we demonstrated that BP1 bound proximally to the Twist promoter and regulated its expression. BP1 induced an EMT in both cell lines as seen by (a) lack of expression of E-cadherin, and an increase of vimentin and fibronectin expression, as well as (b) morphological changes, including a spindle-like, and more migratory phenotype. We also observed that BP1 induced expression of the cytokine IL6 in both cell lines. We conclude that BP1 regulates the EMT in breast cancer cells via the Twist and IL6 pathways. Citation Format: Farhad Vesuna, Bin-Jin Hwang, Jinguen Rheey, Mamta Giri, Mandeep Gill, Sidney W. Fu, Ashley Irving, Ala Lisok, Yehudit Bergman, Venu Raman, Patricia E. Berg. BP1 induces an epithelial to mesenchymal transition in breast cancer cells by modulating the Twist/IL6 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3338.

Abstract 1144: Building Kras (G12D)/Trp53 null/Pdx1-cre (KPC) mouse allograft tumor model for efficacy evaluation of combinatory immunotherapies

Abstract Pancreas ductal adenocarcinoma (PDAC) has dreadful 5-year survival of 6% and remains one of the most difficult to treat cancers with very few standard-of-care options. Fueled by the clinical success of immune checkpoint inhibitors in other solid tumors, several clinical trials have been conducted in PDAC patients. Unfortunately, those attempts have achieved limited clinical benefits as single-agent treatment. Now investigators are eager to test combinatory therapies of chemotherapies, small-molecule immune modulators, and immune checkpoint antibodies. Highly relevant preclinical models are very much needed for proof-of-principle efficacy evaluation. KPC model, first described by Tuveson and colleagues as LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre, and later established in a variety of derivatives by several labs with Trp53 heterozygous or homozygous knockouts, recapitulates human PDAC tumors in many aspects including morphologic small ductal tumors with enriched stromal contents; key features of immune microenvironment of PDAC representing robust inflammatory reactions, like high levels of B cells and macrophages and exclusion of effector T cells; and most importantly, is nonresponsive to a series of chemotherapies and immune checkpoint inhibitors, including PD1 and CTLA4 antibodies. These features make KPC model an ideal model to test combinatory therapies for PDAC. However, parental KPC model is difficult to use for pharmacologic studies due to the spontaneous nature of tumor onset and progression. The compound mutant mice are also costly to breed. We have generated a transplantable KPC tumor model by passaging the primary tumor subcutaneously in the C57BL/6 mice. These KPC allografts retain morphologic similarity to human PDAC. Immune profiling of both subcutaneously and orthotopically grafted tumors demonstrated highly enriched CD45+ lymphocytes, particularly B cells and macrophages, in the tumor. The tumor is also nonresponsive to gemcitabine treatment, in line with the data from parental line. Orthotopically engrafted tumor leads to lethality of the host with the median survival time of 25 days. We are now testing a variety of combinatory therapies with chemotherapies, immune modulators and immune checkpoint antibodies. The results will be presented at the meeting. Citation Format: Yanrui Song, Benqi Liu, Annie Xiaoyu An, Likun Zhang, Jie Cai, Qian Shi, Henry Qixiang Li, Davy Xuesong Ouyang. Building Kras (G12D)/Trp53 null/Pdx1-cre (KPC) mouse allograft tumor model for efficacy evaluation of combinatory immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1144.

PO-427 Establishment of KRAS (G12D)/Trp53 null/Pdx1-cre (KPC) mouse homograft tumour models to facilitate preclinical efficacy evaluation of combinatory immunotherapies

IntroductionPancreatic ductal adenocarcinoma (PDAC) has a poor 5 year survival rate of 6% and remains one of the most difficult to treat cancer types, with very few standard of care options. Fueled by the clinical success of immune checkpoint inhibitors in other solid tumours, several clinical trials have been conducted in PDAC patients. Unfortunately, these attempts have achieved limited clinical benefit as single agent treatments. Now investigators are eager to test combinatory regimens of chemotherapies, small molecule immunomodulators, and immune checkpoint antibodies. Therefore, highly relevant preclinical models are much needed for proof of principle efficacy evaluation studies.KPC models, first described by Tuveson et al;Cancer Res 20166:242–47) such as LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre, and later established as a variety of derivatives with Trp53 heterozygous or homozygous knockouts, recapitulate human PDAC tumours in many aspects including: morphological small ductal tumours with enriched stromal contents, key features of the PDAC immune microenvironment representing robust inflammatory reactions, e.g. high levels of B cells and macrophages, and exclusion of effector T cells. Most importantly, no response to a series of chemotherapies and immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, is observed. These features make KPC models an ideal platform to test combinatory therapies for PDAC within a hostile tumour microenvironment.Material and methodsWe have generated a transplantable KPC tumour model by passaging the primary tumour subcutaneously in C57BL/6 mice. These KPC homografts (KPC MuPrime™) retain morphological similarity to human PDAC.Results and discussionsImmunoprofiling of both subcutaneously and orthotopically grafted tumours demonstrated highly enriched CD45 +lymphocytes, particularly B cells and macrophages, in the tumour. The tumour responded poorly to gemcitabine as well as other CTX and targeted treatments, in line with data from the parental line. Orthotopically engrafted tumours led to lethality of the host with a median survival time of 25 days.ConclusionWe are now testing a variety of combinatory therapies including chemotherapies, immunomodulators, and immune checkpoint antibodies with this model.

Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.

Diagnostic and prognostic value of KRAS mutations in circulating pancreatic ductal adenocarcinoma tumor DNA

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic tumor and one of the most malignant tumors worldwide. Circulating tumor DNA (ctDNA) has significant diagnostic and prognostic value for cancer patients. Methods: Surgical specimens and plasma samples were obtained from a total of 35 patients with PDAC at the Peking University Cancer Hospital between June 2016 and May 2017. To investigate KRAS mutations (G12R, G12V or G12D) in plasma ctDNA, digital polymerase chain reaction (PCR) was performed on samples obtained from PDAC patients before and after surgical resection. Results: KRAS mutations (G12R, G12V or G12D) between surgical tissue DNA (tDNA) and preoperative plasma ctDNA (pre-ctDNA) were consistent in 27 of 35 samples (77.1%, kappa index =0.397, P=0.003). Moreover, pre-ctDNA and postoperative plasma ctDNA (post-ctDNA) showed statistically significant associations with CA19-9 levels before surgery (P=0.027 and P=0.003, respectively). In addition, the Kaplan-Meier univariate and Cox multivariate analysis revealed that pre-ctDNA (G12V), post-ctDNA (G12V), or pre-post ctDNA (G12V, G12D) might be independent prognostic factors for overall survival (OS) and progression-free survival (PFS). Conclusions: Analysis of pre-ctDNA, post-ctDNA, and pre-post ctDNA showed high PDAC diagnostic and prognostic potential in patients.